Monoclonal antibodies to a new antigenic marker in epithelial prostatic cells and serum of prostatic cancer patients

ABSTRACT

Monoclonal antibodies to prostatic cells, are produced by a hybridoma formed by fusing mouse lymphocytes and mouse myeloma cells. The monoclonal antibodies show specificity for a non-soluble, membrane associated, organ specific antigenic determinant limited in its distribution to normal and neoplastic, human prostate epithelial cells. The monoclonal antibodies, specifically 7E11-C5 monoclonal antibodies, may be suitable for diagnostic uses.

TABLE OF CONTENTS

1. Introduction

2. Background of the Invention

2.1. Monoclonal Antibodies

2.2. Application of Monoclonal Antibodies to Cancer

2.3. Monoclonal Antibodies to Prostate Cells

3. Summary of the Invention

4. Description of the Figures

5. Description of the Invention

5.1. The Antigen

5.2. Somatic Cell

5.3. Myeloma Cells

5.4. Fusion

5.5. Isolation of Clones and Antibody Detection

5.6. Cell Propagation and Antibody Production

5.7. In Vitro Diagnostic Uses for Monoclonal Antibodies to HumanProstate Cancer

5.7.1. Immunohistological and Immunocytological Applications

5.7.2. Immunoserological Applications

5.8. In Vivo Diagnostic and Therapeutic Uses for Monoclonal Antibodiesto Human Prostate Cancer

5 8.1. Tumor Localization

5.8.2. Passive Immunotherapy for Treatment of Human Cancer

5.8.3. Treatment of Human Cancer with Monoclonal Antibody Conjugates

6. Examples

6.1. Cell Lines and Tissues

6.2. Immunization and Cell Fusion

6.3. Isolation of Plasma Membrane-Enriched Fraction

6.4. Dot-Immunobinding Assay

6.5. Enzyme Linked Immunosorbent Assay (ELISA)

6.6. Isotyping of Monoclonal Antibodies

6.7. Indirect Immunoperoxidase Staining of Tumor Specimens of MonoclonalAntibodies 7E11C5.

6.8. Competitive Binding ELISA

1. INTRODUCTION

This invention relates to the production of and applications formonoclonal antibodies specific for prostatic tumor antigens. Moreparticularly, this invention relates to monoclonal antibodies againstnon-soluble, membrane associated, organ specific determinants expressedmaximally on human normal and neoplastic prostatic epithelium.Monoclonal antibodies capable of reacting with membrane associatedsurface antigens are of value for the immuno-classification anddetection of disease and represent novel agents for immunotherapy. Themonoclonal antibodies of this invention possess distinctivecharacteristics and capabilities which make them suitable for in vitroclinical diagnostic and prognostic purposes. Moreover, they are of greatpotential importance for in vivo tumor localization and cancer therapyin humans.

The monoclonal antibodies exhibit a high level of binding to humanprostatic cancer cells and normal prostatic epithelium and arepotentially capable of experimental in vivo tumor localization. Theybind to well-differentiated as well as to poorly-differentiated tumors.

The invention provides methods for production of the monoclonalantibodies by hybridoma techniques. Once cloned, cell lines can bemaintained continuously to produce an unlimited homogeneous monoclonalantibody population that can be isolated and/or purified and usedclinically for in vitro immunohistological, immuno-cytological orimmuno-serological diagnosis, in vivo diagnosis by localization oftumors and metastases, and immunotherapy of human cancers, particularlythose of the prostate.

2. BACKGROUND OF THE INVENTION

2.1. Monoclonal Antibodies

Kohler and Milstein are generally credited with having devised thetechniques that successfully resulted in the formation of the firstmonoclonal antibody-producing hybridomas [Kohler, G. and Milstein, C.,Nature 256:495-497 (1975); Eur. J. Immunol. 6:511-519 (1976)]. By fusingantibody-forming cells (spleen lymphocytes) with myeloma cells(malignant cells of bone marrow primary tumors) they created a hybridcell line, arising from a single fused cell hybrid (called a hybridomaor clone) which had inherited certain characteristics of both thelymphocytes and myeloma cell lines. Like the lymphocytes (taken fromanimals primed with sheep red blood cells as antigen), the hybridomassecreted a single type of immunoglobulin specific to the antigen;moreover, like the myeloma cells, the hybrid cells had the potential forindefinite cell division. The combination of these two features offereddistinct advantages over conventional antisera. Whereas antisera derivedfrom vaccinated animals are variable mixtures of polyclonal antibodieswhich never can be reproduced identically, monoclonal antibodies arehighly specific immunoglobulins of a single type. The single type ofimmunoglobulin secreted by a hybridoma is specific to one and only oneantigenic determinant, or epitope, on the antigen, a complex moleculehaving a multiplicity of antigenic determinants. For instance, if theantigen is a protein, an antigenic determinant may be one of the manypeptide sequences [generally 6-7 amino acids in length (Atassi, M. Z.,Molec. Cell. Biochem. 32:21-43 (1980))] within the entire proteinmolecule. Hence, monoclonal antibodies raised against a single antigenmay be distinct from each other depending on the determinant thatinduced their formation; but for any given clone, all of the antibodiesit produces are identical. Furthermore, the hybridoma cell line can bereproduced indefinitely, is easily propagated in vitro or in vivo, andyields monoclonal antibodies in extremely high concentration.

2.2. APPLICATION OF MONOCLONAL ANTIBODIES TO CANCER

Monoclonal antibodies produced by hybridoma technology are potentiallypowerful tools for cancer detection, diagnosis and therapy. [For ageneral discussion of the topic, see Hybridomas in Cancer Diagnosis andTreatment, Mitchell, M. S. and Oettgen, H. F., (eds.), Progress inCancer Research and Therapy, Vol. 21, Raven Press, New York (1982)]. Ithas been reported that monoclonal antibodies have been raised againsttumor cells [U.S. Pat. No. 4,196,265], carcinoembryonic antigen [U.S.Pat. No. 4,349,528], and thymocytes, prothymocytes, monocytes andsuppressor T cells [U.S. Pats. Nos. 4,364,933; 4,364,935; 4,364,934;4,364,936; 4,364,937; and 4,364,932]. Recent reports have demonstratedthe production of monoclonal antibodies with various degrees ofspecificity to several human malignancies, including prostaticcarcinomas [Webb, K. et al., Canc. Immunol. Immunoth. 17:7-17 (1984),Carrol. A., Clin. Immun. and Immunopath. 33:268-281 (1984)], mammarytumor cells [Colcher, D. et al., Proc. Natl. Acad. Sci. U.S.A.78:3199-3203 (1981)], lung cancers [Cuttitta, F., et al., Proc. Natl.Acad. Sci: U.S.A. 78:495-4595 91981 )] malignant melanoma [Dippold, W.G. et al., Proc. Natl. Acad. Sci. U.S.A., 77:6114-6118 (1980)],colorectal carcinoma [Herlyn, M. et al., Proc. Natl. Acad. Sci. U.S.A.76:1438-1442 (1979)], lymphoma [Nadler, L. M. et al., J. Immunol.125:570-577 (1980)], and neuroectodermal tumors [Wikstrand, C. J. andBigner, D. C., Cancer Res. 42:267-275 (1982].

Investigators have noted the potential immuno-therapeutic value ofmonoclonal antibodies and some investigators have investigatedtherapeutic efficacy in both animal and human subjects [Oldham, R. K.,J. Clin. Oncol., 1:582-590 (1983); Miller, R. A. et al., Blood,62:988-995 (1983); Miller R. A. et al., New Engl. J. Med. 306:517-522(1982); Ritz, J. and Schlossman, S., Blood, 59:1-11 (1982); and Kirch,M. E. and Ulrich, H. J. Immunol. 127:805-810 (1981)]. Although moststudies have described the effects of cytotoxic drug-antibodyconjugates, [Beverly, P. C. L., Nature, 297:358-9 (1982); Krolick, K. A.et al., Nature, 295:604-5 (1982); Krolick, K. A. et al., Proc. Natl.Acad. Sci. U.S.A., 77:5419-23 (1980); Arnon, R. and Sela, M., Immunol.Rev., 62:5-27 (1982); Raso, V. et al., Cancer Res., 42:457-64 (1982);and DeWeger, R. A. and Dullens, H. F. J., Immunol. Rev. 62:29-45(1982)], experimental and clinical studies on passive immunotherapieshave been attempted Sears, H. F. et al., Lancet, i:762-65 (1982);Rosenberg, S. A. and Terry, W. D., Cancer Res., 25:323-88 (1977);Herlyn, D. M. et al., Cancer Res., 40:717-21 (1980); Scheinberg, D. A.and Strand, M., Cancer Res. 42:44-9 (1982); Koprowski, H. et al., Proc.Natl. Acad. Sci. U.S.A., 75:3405-9 (1978); and Young, Jr., W. W. andHakomori, S. I., Science, Science, 211:487-9 (1981)]. In theexperimental setting, however, most studies have dealt with theconcurrent administration of monoclonal antibody and tumor inoculum, oradministration within several days of the implantation of tumor cells,resulting in a decreased tumor take or growth rate or xenografts. Inthis context, these data form a basis for the immunoprophylaxis of tumordevelopment.

It is an object of the present invention to provide monoclonalantibodies having a broad range of utilities in both therapy anddiagnostics of prostate cancer.

2.3. MONOCLONAL ANTIBODIES TO PROSTATE CELLS

Prostatic epithelium has limited distribution, does not carry outfunctions vital for the survival of a patient, and was shown to produceorgan specific, albeit secretory, macromolecules. Cancer of the prostateis the second most frequent tumor of males in the United States. Unknownetiology, variable pathology, intricate relationship to endocrinefactors, and anaplastic progression contribute to the complexity of thisdisease and limited effectiveness of available therapies. The progresstoward establishing effective immunological methods for early detectionand successful management of cancer of the prostate (hereinafterreferred to as CaP) may depend on laboratory experimentation with mostsuitable models used as reagents for monoclonal antibody production.Similarly to other tumors, prostate cancer specific antigen has not beendefined by monoclonal antibodies, although some CaP-associated epitopeswere already identified, and will be reviewed below.

Several investigators have reported on the production of monoclonalantibodies against epitopes of various normal and malignant prostatecell components. [Carroll, A. et al., Clin. Immunol. Immunopath.33:268-281 (1984); Webb, K. et al., Cancer Immunol. Immunoth. 17:7-17(1984); Frankel, A. et al., Proc.Natl. Acad. Sci. U.S.A. 79:903-907(1982); Frankel, A. et al., Cancer Res. 42:3714 (1982); Starling, J. etal., Cancer Res. 42:3084 (1982)].

Several monoclonal antibodies are available against two wellcharacterized, purified to homogeneity, soluble glycoproteins producedand secreted by either normal or malignant human prostatic epithelium.Wang et al. described a human prostate specific antigen (P.S.A.) thathas a molecular weight of 34,000, and is present in human prostate,seminal plasma and CaP cells. Readily produced polyclonal and monoclonalantibodies to purified P.S.A. established this antigen as aserodiagnostic marker for CaP, a marker for human prostatic epithelialcells and an immunohistological marker for prostate neoplasms. Anotherstrictly organ specific, well known marker protein of normal andneoplastic human prostatic cell is human prostatic acid phosphatase(PAP). PAP is a glycoprotein with molecular weight of 100,000 in whichthe aminoterminal sequence and carbohydrate composition has beenestablished. Murine monoclonal antibodies identify 3 distinct antigenicdeterminants and several sensitive immunoassays to measure PAP weredeveloped. Preliminary experiments suggest that monoclonal anti-PAPantibody has potential for antibody-directed radioimaging and monoclonalantibody targeted chemotherapy of prostate cancer. Both P.S.A. and PAPare secretory products of diagnostic value and can be detected, not onlyin cells, but also in plasma of patients with advanced CaP, nude micebearing LNCaP tumors (derived from a metastic lesion of human prostaticcarcinoma) and culture supernatants from malignant human prostatic cellsexpressing this marker. Such distribution of soluble antigens couldimpair the monoclonal antibodies in reaching their ultimate target--themalignant cell.

Another strategy in monoclonal antibody production against humanprostatic cancer cells has been the utilization as immunogens of wholecells or fractionated cell preparations from established in vitrocultures of human malignant prostatic cells PC-3 [Kaighn, M. et al.,Invest. Urol. 17:16 (1979)] and DU-145 [Stone, K. et al., Int. J.Cancer21:274 (1978)]. Neither of these cells lines, however, maintain a fullset of characteristic biological or biochemical markers characteristicof prostatic epithelium and malignant prostatic cells, e.g., productionof secretory human prostatic acid phosphatase, organ specific prostateantigen, responsiveness to androgens or the presence of the Ychromosome. Such cells may not be optimally representative of themajority of prostatic tumors as seen by the clinician and pathologist. Avariety of monoclonal antibodies selected for in depth studies by othershave shown reactivity not only with cell surface or cytoplasmic antigensof prostate cancer cells, but also with cells for other malignancies andsignificantly, with non-prostatic normal human tissues.

Starling, J. et al. (Canc. Res. 42:3084-3089 (1982) reported that ashort immunization schedule with DU-145 cells (days 0, 8 and 15) ofBALB/c mice resulted in isolation of monoclonal antibody 83.21 whichbound to surfaces of DU-145 cells. This monoclonal antibody was of theIgM class and did not bind to a variety of human tumors, normal tissues,several cell lines, nor to normal human prostatic epithelium. Membranepreparations from one metastatic CaP, PC-3 and DU-145 cells efficientlybound monoclonal antibody 83.21. The spectrum and degree of reactivitiesof this monoclonal antibody with different cells varied depending uponthe antibody binding assays used such as immunofluorescence, complementdependent cytotoxicity or quantitative adsorption analysis. Epitopesdetected by monoclonal antibody 83.21 were present on 58% primary CaPand, 17% CaP metastases. In addition significant cross-reactions withtransitional cell carcinoma of the bladder, cytomegalovirus transformedhuman embryonic cell line as well as with proximal convoluted tubules ofnormal kidney were seen.

Wright, G. et al. [Canc. Res. 53:5509-5516 (1983)] immunizing mice withPC-3 cells obtained monoclonal antibody P 6.2. This antibody, also ofthe IgM class, reacted with 72% of paraffin embedded specimens of CaP,however lung cancer, breast cancer, pancreas cancer and human normalkidney also stained.

Ware, J. et al. [Canc. Res. 42:1215-1222 (1982)] produced monoclonalantibody alpha Pro-3 of the IgG2a sub-class by immunizing mice with PC-3cells. This antibody recognized an antigen (p54) concentrated in humanprimary prostatic carcinoma removed surgically. The antigen p54 is alsopresent in variable amounts in extracts from benign prostatichypertrophy (BPH), testicular tumors, kidney cancer, thyroid cancer,bladder cancer, ovarian cancer and in normal non-prostatic tissues.Cultured cells such as PC-3, human breast carcinoma (MDA-MB-231) andnormal human fibroblasts (IMR90) all show surface binding of monoclonalantibody Pro-3. Binding of alpha Pro-3 to another CaP derived cell line,DU-145 is only minimal. Another epitope of the p54 antigen is alsorecognized by monoclonal antibody alpha Pro-5.

Short term immunization with a mixture of three cell lines derived fromhuman CaP led to the isolation of monoclonal antibody alpha Pro-13 byWebb, K. et al. [Canc. Immunol. Immunother. 17:7-17 (1984)]. In thesolid phase binding assay this antibody reacted with 8 out of 10extracts of CaP and benign prostate hypertrophy, but bladder cancer andkidney cancers were also positive, though to a smaller degree.Immunoperoxidase staining of frozen sections produced positive stainingin epithelial cells in 4 out of 12 CaP and in 1 out of 6 BPH specimens.Cross-reactivity with non-prostatic tissue occurred with renalcarcinoma, glands of the normal trachea, and vessel endothelium fromtestis and tonsils. Among cultured cells, PC-3, lung cancer, coloncancer and melanoma contained surface molecules recognized by alphaPro-13. The antigen defined by monoclonal antibody alpha Pro-13 is aglycoprotein of 120,000 molecular weight (nonreduced) which isintrinsically stable on the cell surface with negligible release in cellculture supernatant or solubilized significantly only after CHAPS, butnot Triton X-100 detergent treatment.

Several monoclonal antibodies reactive with surface components of normaland malignant human prostatic epithelium were obtained by Frankel, A.[Proc. Natl. Acad. Sci. USA, 79:903-907 (1982)] who used membraneenriched fractions from benign prostatic hypertrophy for immunization.All of them exhibited significant cross-reactivity with either kidney,spleen, thymus, pancreas, bladder, lung thyroid or brain tissue.

Carroll, A. et al. [Clin. Immunol, Immunopath. 33:268-281 (1984)] raisedmonoclonal antibodies to PC-3 cells. One hybridoma--F77-129 (IgG3subclass) reacted with prostatic cancer cell lines (PC-3 and DU-145), 3out of 4 breast cancer cell lines and one colon cancer line.Immunoperoxidase staining of human tissues confirmed binding to normaland malignant human prosatic and breast tissue. Radioiodinated F77-129localized readily in tumors induced by injection of PC-3 cells into nudemice.

There is a need for monoclonal antibodies which are prostate specificand which will not cross react with other tissue types. The use of suchantibodies can add significant information regarding functionalclassifications of individual prostate tumors to augment clinicalclassifications.

The pattern of staining for the monoclonal antibody of this invention isdistinct from the reactivities of previously described monoclonalantibodies which recognize antigens expressed by prostate tumors. Themonoclonal antibodies provided herein stain malignant prostateepithelial cells intensely, mostly on the periphery of cells with asmall degree of heterogeneity. Normal prostate epithelial cells orbenign prostatic hypertrophy cells showed either faint or only amoderate degree of staining. By comparison with the monoclonalantibodies described by Frankel (supra), the monoclonal antibodies ofthe invention do not stain non-epithelial components of the prostate(fibers, muscle, stroma, vessels, etc.). Additionally no specificstaining was observed in non-prostatic malignant tumors.

3. SUMMARY OF THE INVENTION

Prior to the present invention, applicant believes that a non-soluble,prostate cancer specific antigen has not been defined by monoclonalantibodies, although some cancer of the prostate-associated epitopeshave been identified.

The present invention provides methods and compositions for producingnovel monoclonal anti-prostate carcinoma antibodies with specificbinding capabilities and encompasses the used said antibodies for cancerimmunodiagnosis, prognosis and therapy in humans. Specifically, theinvention provides novel hybridoma-derived monoclonal antibodies whichdemonstrate a narrow spectrum of organ-specific reactivity withnon-soluble, membrane associated antigenic determinants (epitopes)present on normal neoplastic and malignant human prostatic epithelium.The monoclonal antibodies do not react specifically with non-prostatictumors and other tissues. The monoclonal antibodies stain malignantprostatic cells intensely and non-malignant prostatic epithelium weakly.

In addition, to their use as in vitro immuno-histological reagents forcancer diagnosis, the present invention contemplates the use of themonoclonal antibodies for in vivo diagnosis. Because of their ability totarget prostate carcinoma cells, the monoclonal antibodies can be usedin tumor localization and in the monitoring of metastases.

The invention further contemplates the use of the monoclonal antibodiesprovided herein as a diagnostic and prognostic tool for detection ofcancer of the prostate. The monoclonal antibodies provided herein can beused as in vitro immunoserological and immunocytological reagents onbody fluids to detect the presence of the specific antigen and/or cellsbearing antigen. The monoclonal antibodies thereby permit non-invasivediagnosis of prostate carcinomas and place the clinician in a betterposition to diagnose, monitor and treat prostate cancer.

The monoclonals of the present invention may also be useful in thedetection of anti-idiotypic antibodies.

The present invention provides methodologies useful in research for theevaluation of parameters associated with the use of monoclonalantibodies against human tumors in passive human immunotherapy. Themonoclonal antibodies can be used as probes to investigate the roles ofantigen density, tumor growth rates, tumor size, cellular heterogeneityand other variables in the susceptibility of tumors to immunotherapy.The monoclonal antibodies may also induce a modification of the hostanti-tumor immune response through the formation of anti-idiotypicantibodies to immune monoclonal antibodies.

The invention contemplates the use of the antibodies provided herein incovalent combination with radioactive, cytotoxic or chemotherapeuticmolecules. For instance, the monoclonal antibodies can be conjugated tocertain cytotoxic compounds, including, but not limited to, radioactivecompounds, diphtheria toxin (chain A), ricin toxin (chain A),adriamycin, chlorambucil, daunorubicin, or pokeweed antiviral protein toenhance their tumoricidal effectiveness.

The present invention further contemplates the use of monoclonalantibodies in immunoadsorption procedures to effectively separateprostate cancer cells from marrow elements based upon antibody binding,and in procedures to eliminate malignant cells while sparing bone marrowcells. Because such procedures require a tumor antigen, such as the onerecognized by the monoclonal antibodies of this invention, which is notfound on bone marrow stem cells or lymphoid cells, the monoclonalantibodies represent a reagent useful for eliminating disseminatedprostate cancer cells from autologous bone marrow.

Because the monoclonal antibodies are produced by hydridoma techniques,the present invention provides theoretically immortal cell lines capableof consistently producing high titers of single specific antibodiesagainst a distinct prostate carcinoma antigen. This is a distinctadvantage over the traditional technique of raising antibodies inimmunized animals where the resulting sera contain multiple antibodiesof different specificities that vary in both type and titer with eachanimal, and, in individual animals, with each immunization.

4. DESCRIPTION OF THE FIGURES

FIG. 1 outlines the procedure used in preparing the LNCaP cell fractionused in immunization for monoclonal antibody production.

FIG. 2 is a photograph showing the indirect immunoperoxidase staining ofLNCaP cells with monoclonal antibody 7E11-C5.

FIG. 3 is a photograph showing indirect immunoperoxidase staining ofLNCaP cells with monoclonal antibody 9H10-A4.

5. DESCRIPTION OF THE INVENTION 5.1. The Antigen

In the embodiment of the invention described in the Examples whichfollow, LNCaP prostate carcinoma cells and partially purified LNCaPplasma membranes were used as "antigen" or immunogen. As demonstrated byexperiments described below, the epitope recognized by the antibody ofthis invention is present in several surgery and autopsy specimens fromlocalized and metastatic prostate cancer, benign prostatic hypertrophy,normal prostates, and prostatic cultured cell lines. Hence, such cellsexpressing organ (or tissue) specific antigens also represent potential"antigen" or sources of antigen with which to immunize animals to obtainsomatic cells for fusion.

5.2. SOMATIC CELLS

Somatic cells with the potential for producing antibody and, inparticular B lymphocytes, are suitable for fusion with a B-cell myelomaline. Those antibody-producing cells that are in the dividingplasmablast stage fuse preferentially. Somatic cells may be derived fromthe lymph nodes, spleens and peripheral blood of primed animals and thelymphatic cells of choice depending to a large extent on their empiricalusefulness in the particular fusion system. Once-primed orhyperimmunized animals can be used as a source of antibody-producinglymphocytes. Mouse lymphocytes give a higher percentage of stablefusions with the mouse myeloma lines described in section 5.3. However,the use of rat, rabbit, and frog cells is also possible.

Alternatively, human somatic cells capable of producing antibody,specifically B lymphocytes, are suitable for fusion with myeloma celllines. While B lymphocytes from biopsied spleens or lymph nodes ofindividual may be used, the more easily accessible peripheral blood Blymphocytes are preferred. The lymphocytes may be derived from patientswith diagnosed carcinomas.

5.3. MYELOMA CELLS

Specialized myeloma cell lines have been developed from lymphocytetumors for use in hybridoma-producing fusion procedures [Kohler, G. andMilstein, C., Eur. J. Immunol. 6:511-519 (1976); M. Schulman et al.,Nature 276: 269-270 (1978)]. The cell lines have been developed for atleast three reasons. The first is to facilitate the selection of fusedhybridomas among unfused and similarly indefinitely self-propogatingmyeloma cells. Usually, this is accomplished by using myelomas withenzyme deficiencies that render them incapable of growing in certainselective media that support the growth of hybridomas. The second reasonarises from the inherent ability of lymphocyte tumor cells to producetheir own antibodies. The purpose of using mono-clonal techniques is toobtain immortal fused hybrid cell lines that produce the desired singlespecific antibody genetically directed by the somatic cell component ofthe hybridoma. To eliminate the production of tumor cell antibodies bythe hybridomas, myeloma cell lines incapable of producing light or heavyimmunoglobulin chains or those deficient in antibody secretionmechanisms are used. A third reason for selection of these cell lines isfor their suitability and efficiency for fusion.

Several myeloma cell lines may be used for the production of fused cellhybrids, including X63-Ag8, NSI-Ag4/1, MPC11-45.6TG1.7, C63-Ag8.653,Sp2/0-Ag14, FO, and S194/5XX0.BU.1, all derived from mice,210.RCY3.Agl.2.3 derived from rats and U-226AR, and GM1500GTGAL2,derived from rats and U-226AR, and GM1500GTGAL2, derived from humans,[G. J. Hammerling, U. Hammerling and J. F. Kearney (editors), MonoclonalAntibodies and T-cell Hybridomas IN: J. L. Turk (editor) ResearchMonographs in Immunology, Vol. 3, Elsevier/North Holland BiomedicalPress, New York (1981)].

5.4. FUSION

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion as in the example in Section 6.2.(though the proportion may vary from about 20:1 to about 1:1),respectively, in the presence of an agent or agents (chemical orelectrical) that promote the fusion of cell membranes. It is oftenpreferred that the same species of animal serve as the source of thesomatic and myeloma cells used in the fusion procedure. Fusion methodshave been described by Kohler and Milstein [Nature 256:495-497 (1975)and Eur. J. Immunol. 6:511-519 (1976)], and by Gefter et al. [SomaticCell Genet. 3:231-236 1977)]. The fusion-promotion agent used by thoseinvestigators were Sendai virus and polyethylene glycol (PEG),respectively. The fusion procedure of the example of the presentinvention is a modification of the method of Kohler and Milstein, supra.

5.5. ISOLATION OF CLONES AND ANTIBODY DETECTION

Fusion procedures usually produce viable hybrids at very low frequency,about 1×10⁻⁶ to 1×10⁻⁸. Because of the low frequency of obtaining viablehybrids, it is essential to have a means to select fused cell hybridsfrom the remaining unfused cells, particularly the unfused myelomacells. A means of detecting the desired antibody-producing hybridomasamong the other resulting fused cell hybrids is also necessary.

Generally, the fused cells are cultured in selective media, for instanceHAT medium containing hypoxanthine, aminopterin and thymidine. HATmedium permits the proliferation of hybrid cells and prevents growth ofunfused myeloma cells which normally would continue to divideindefinitely. Aminopterin blocks de novo purine and pyrimidine synthesisby inhibiting the production of tetrahydrofolate. The addition ofthymidine bypasses the block in pyrimidine synthesis, while hypoxanthineis included in the media so that inhibited cells synthesize purine usingthe nucleotide salvage pathway. The myeloma cells employed are mutantslacking hypoxanthine phosphoribosyl transferase (HPRT) and thus cannotutilize the salvage pathway. In the surviving hybrid, the B lymphocytesupplies genetic information for production of this enzyme. Since Blymphocytes themselves have a limited life span in culture(approximately two weeks), the only cells which can proliferate in HATmedia are hybrids formed from myeloma and spleen cells.

To facilitate screening of antibody secreted by the hybrids and toprevent individual hybrids from overgrowing others, the mixture of fusedmyeloma and B lymphocytes is diluted in HAT medium and cultured inmultiple wells of microtiter plates. In two to three weeks, when hybridclones become visible microscopically, the supernatant fluid of theindividual wells containing hybrid clones is assayed for specificantibody. The assay must be sensitive, simple and rapid. Assaytechniques include radioimmunoassays, enzyme immunoassays, cytotoxicityassays, plaque assays, dot immunobinding assays, and the like.

5.6. CELL PROPAGATION AND ANTIBODY PRODUCTION

Once the desired fused cell hybrids have been selected and cloned intoindividual antibody-producing cell lines, each cell line may bepropagated in either of two standard ways. A sample of the hybridoma canbe injected into a histocompatible animal of the type that was used toprovide the somatic and myeloma cells for the original fusion. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can be tapped to providemonoclonal antibodies in high concentration. Alternatively, theindividual cell lines may be propogated in vitro in laboratory culturevessels; the culture medium, also containing high concentrations of asingle specific monoclonal antibody, can be harvested by decantation,filtration or centrifugation.

5.7. IN VITRO DIAGNOSTIC USES FOR MONOCLONAL ANTIBODIES TO HUMANPROSTATE CANCER 5.7.1. Immunohistological and ImmuncytologicalApplications

The monoclonal antibodies of this invention can be used as probes indetecting discrete antigens in human tumors. The expression or lack ofexpression of these antigens can provide clinically exploitableinformation which is not apparent after standard histopathologicalevaluations. It may thus be possible to correlate the immuno-phenotypesof individual tumors with various aspects of tumor behavior andresponsiveness to certain types of therapies, thus establishingimportant classifications of prognosis.

Monoclonal antibodies produced by the hybridoma methodologies hereindescribed can be used to detect potential prostate carcinoma cells inhistological and cytological specimens and in particular, to distinguishmalignant from non-malignant tumors based on staining patterns andintensities. For instance, using the immunoperoxidase staining techniquedescribed in section 6.7., it has been observed that the monoclonalantibodies of its invention stained neoplastic prostate cells, mostly onthe periphery of cells, with a small degree of heterogeneity.Morphologically nonmalignant prostatic ductal epithelium from benignprostatic hypertrophy and normal prostates generally exhibited a reduceddegree of staining with cellular localization similar to prostaticcancer cells. Staining was completely absent from non-epithelialcomponents of the prostate; no specific staining was observed innon-prostatic malignant tissues nor in normal human organs and tissuesexamined.

As an alternative to immunoperoxidase staining, immunofluorescenttechniques can be used to examine human specimens with monoclonalantibodies to prostate carcinoma. In a typical protocol, slidescontaining cryostat sections of frozen, unfixed tissue biopsy samples orcytological smears are air dried formalin fixed and incubated with themonoclonal antibody preparation ln a humidified chamber at roomtemperature.

The slides are then layered with a preparation of antibody directedagainst the monoclonal antibody, usually some type of anti-mouseimmunoglobulin if the monoclonal antibodies used are derived from thefusion of a mouse spleen lymphocyte and a mouse myeloma cell line. Thisantimouse immunoglobulin is tagged with a compound that fluoresces at aparticular wavelength for instance rhodamine or fluoresceinisothiocyanate. The staining pattern and intensities within the sampleare then determined by fluorescent light microscopy and optionallyphotographically recorded.

5.7 2. IMMUNOSEROLOGICAL APPLICATIONS

The use of the monoclonal antibodies described herein can be extended tothe screening of human biological fluids for the presence of thespecific antigenic determinant recognized. In vitro immunoserologicalevaluation of sera withdrawn from patients thereby permits non-invasivediagnosis of cancers. By way of illustration, human fluids, such aspleural fluids or lymph, can be taken from a patient and assayed for thespecific epitope, either as released antigen or membrane-bound on cellsin the sample fluid, using the anti-prostate carcinoma monoclonalantibodies in standard radioimmunoassays or enzyme-linked immunoassaysknown in the art or competitive binding enzyme-linked immunoassays.

5.8. IN VIVO DIAGNOSTIC AND THERAPEUTIC USES FOR MONOCLONAL ANTIBODIESTO HUMAN PROSTATE CANCER 5.8.1. Tumor Localization

The monoclonal antibodies of this invention are potentially useful fortargeting prostate carcinoma cells in vivo. They can therefore be usedin humans for tumor localization and for monitoring of metastases. Forthis application, it is preferable to use purified monoclonalantibodies. Purification of monoclonal antibodies for humanadministration by ammonium sulfate or sodium sulfate precipitationfollowed by dialysis against saline and filtration sterilization hasbeen described by Miller et al. [IN: Hybridomas in Cancer Diagnosis andTherapy, (1982) supra, p. 134,] and by Dillman et al. [Id. p.155] whichare hereby incorporated by reference. Alternatively, immuno-affinitychromatography techniques may be used to purify the monoclonalantibodies.

The purified monoclonal antibodies can be labeled with radioactivecompounds, for instance, radioactive iodine, and administered to apatient intravenously. After localization of the antibodies at the tumoror metastatic site, they can be detected by emission tomographical andradionuclear scanning techniques thereby pinpointing the location of thecancer. Experimental radioimmunodetection with monoclonal antibodies byexternal scintigraphy has been reported by Solter et al. [Id., p.241]hereby incorporated by reference.

5.8.2. PASSIVE IMMUNOTHERAPY FOR TREATMENT OF HUMAN CANCER

Passive monoclonal serotherapy may be a potential use for the monoclonalantibodies of this invention. By way of illustration, purifiedanti-prostate carcinoma monoclonal antibody (see Section 5.8.1) isdissolved in an appropriate carrier, e.g., saline, with or without humanalbumin, at an appropriate dosage and is administered to a patient. Themonoclonal antibodies are preferably administered intravenously, e.g.,by continuous intravenous infusion over several hours, as in Miller etal., incorporated by reference, supra. Infusions can be administeredover a period of weeks during which the anti-tumor effects aremonitored.

In an alternate embodiment, the antibodies described herein are used tostimulate the production of corresponding anti-idiotypic antibodies. Theexperimental results observed in connection with this invention, i.e.,the high percentage of binding of monoclonal antibody 7E11-C5 detectedin a competitive inhibition ELISA, suggest that sera from prostaticcancer patients contain anti-idiotypic antibodies. In brief,anti-idiotypic antibodies, or antiidiotypes are antibodies directedagainst the antigen combining region or variable region (idiotype) ofanother antibody. In theory, based on Jerne's network model of idiotypicrelationships (Jerne, Ann. Immunol. 125:373, 1974; Jerne et al., EMBO1:234, 1982), immunization with an antibody molecule expressing aparatope (antigen-combining site) for a given antigen should produce agroup of anti-antibodies, some of which share with the antigen acomplementary structure to the paratope. Immunization with asubpopulation of antiidiotypic antibodies should in turn produce asubpopulation of antiidiotypic antibodies which bind the initialantigen. Thus, the administration of the monoclonal antibodies of thepresent invention may result in a modification of the host's anti-tumorimmune response, as the consequence of the formation of anti-idiotypicantibodies which may develop during therapy with the monoclonals.

5.8.3. TREATMENT OF HUMAN CANCER WITH MONOCLONAL ANTIBODY CONJUGATES

The monoclonal antibodies of this invention can be used in conjunctionwith a broad spectrum of pharmaceutical or cytotoxic agents such as:radioactive compounds (e.g., In¹¹¹, I¹²⁵, I¹³¹); agents which bind DNA,for instance, alkylating agents or various antibiotics e.g., daunomycin,adriamycin, chlorambucil); antimetabolites such as methotrexate; agentswhich act on cell surfaces (e.g., venom phospholipases and microbialtoxins); and protein synthesis inhibitors (e.g., diphteria toxin andtoxic plant protein; enzymes that inhibit the action of eukaryoticribosomes (e.g. ricin, ricin A chain and pokeweed antiviral protein.[For reviews on the subject, see Bale et al., Cancer Research40:2965-2972 (1980); Ghose and Blair, J.. Natl. Cancer Inst. 61(3):657-676 (1978); Gregoriadis, Nature 265: 407-411(1978);Gregoriadias, Pharmac. Ther. 10:103-108(1980); Trouet et al., RecentResults Cancer Res. 75:229-235 (1989)]. Of particular importance arethose agents capable of exerting toxic effects at the level of the cellsurface, such as adriamycin [Tritton, T. R. and Yee, G., Science,217:248-50 (1982 )].

The methods used for binding the cytotoxic agents to the monoclonalantibody molecule can involve either non-covalent or covalent linkages.Since non-covalent bonds are more likely to be broken before theantibody complex reaches the target site, covalent linkages are,preferred. For instance, carbodiimide can be used to link carboxy groupsof the pharmaceutical agent to amino groups of the antibody molecule.Bifunctional agents such as dialdehydes or imidoesters can be used tolink the amino group of a drug to amino groups of the antibody molecule.The Schiff base reaction can be used to link drugs to antibodymolecules. This method involves the periodate oxidation of a drug orcytotoxic agent that contains a glycol or hydroxy group, thus forming analdehyde which is then reacted with the antibody molecule. Attachmentoccurs via formation of a Schiff base with amino groups of the antibodymolecule. Additionally, drugs with reactive sulfhydryl groups have beencoupled to antibody molecules.

Similarly, glycosidic enzymes such as neuraminidase or α-mannosidase canbe conjugated to the monoclonal antibodies. Conjugated antibodies can beadministered to patients to achieve enhanced tumoricidal effects throughthe cytotoxic action of the chemotherapeutic agents or the increasedbinding effect of the glycosidic enzymes.

6. EXAMPLES 6.1. Cell Lines and Tissues

The LNCaP cell line was established from a metastatic lesion of humanprostatic carcinoma. The LNCaP cells grow readily in vitro (up to 8×10⁵cells/ cm² ; doubling time, 60 hours), form clones in semisolid media,and show an aneuploid (modal number, 76 to 91) human male karyotype withseveral marker chromosomes. The malignant properties of LNCaP cells aremaintained. Athymic nude mice develop tumors at the injection site(volume-doubling time, 86 hours). Functional differentiation ispreserved; both cultures and tumor produce a prostate acid phosphatase(PAP) and prostate specific antigen (P.S.A.). High-affinity specificandrogen receptors are present in the cytosol and nuclear fractions ofcells in culture and in tumors. Estrogen receptors are demonstrable inthe cytosol. The model is hormonally responsive. In vitro,α-dihydro-testosterone modulates cell growth and stimulates acidphosphatase production. In vivo, the frequency of tumor development andthe mean time of tumor appearance are significantly different for eithersex. LNCaP cells, therefore, meet criteria of a versatile model forimmunological studies of human prostatic cancer in the laboratory.

Seven malignant cell lines of human origin were obtained from J. Fogh ofMemorial Sloan-Kettering Institute and included: DU-145 and PC-3 derivedfrom prostatic cancer; MCF-7, derived from pleural effusion of scirrhouscarcinoma of the breast [Soule, D. G. et al., J. Natl. Cancer Inst.,51:1409-1416 (1973)]; MeWo, malignant melanoma; RT-4, transitional cellcarcinoma; HT-29, adenoma of the colon and A209, rhabdomyosarcoma. Fourother cell lines (two malignant and two normal), isolated andestablished at Roswell Park Memorial Institute were also used: TT,thyroid medullary carcinoma, pancreatic cancer, BG-9 and MLD--bothnormal diploid neonatal foreskin fibroblast (see Horoszewicz et al.,Infect. Immun. 19:720-726, 1978; Chen et al., Human PancreaticAdenocarcinoma, Vol. 18: 24-32, 1982; Leong et al., Advances in ThyroidNeoplasia 1984:95-108, 1982). All of the above cell lines were routinelymaintained in RPMI medium 1640 (Roswell Park Memorial Institute,Buffalo, N.Y.) supplemented with 10% heat inactivated fetal bovineserum, 1 mM L-glutamine, and 50 μg/ml of penicillin and streptomycin(Gibco, Grand Island, N.Y.).

Fresh normal, benign and malignant prostate cancer tissues were obtainedeither from the Department of Surgery or the Department of Pathology atRoswell Park Memorial Institute. The tissues were quick frozen in M-1embedding matrix (Lipshaw Corp., Detroit, Mich.) and stored at -80° C.

6.2. IMMUNIZATION AND CELL FUSION

Ten week old male Balb/c mice (West Seneca Laboratory, West Seneca,N.Y.) received intraperitoneal injections (2×10⁷ cells/0.2 ml) of washed(3 times in RPMI medium 1640, Roswell Park Memorial Institute, Buffalo,N.Y.) live LNCaP cells suspended in RPMI medium 1640, at monthlyintervals for 3 months. Three days before fusion, the mice received anintraperitoneal challenge of 2×10⁷ cells in RPMI medium 1640 and anintravenous injection of the plasma membrane isolated from 1×10⁸ LNCaPcells. Cell fusion was carried out using a modification of the proceduredeveloped by Kohler and Milstein [Nature (Lond.) 256:495-497 (1975)].Mouse splenocytes (1×10⁸ cells) were fused in HyBRL-Prep 50%polyethylene glycol 1450, Bethesda Research Laboratories, Inc.,Gaithersburg, Md.) with 5×10⁷ mouse myeloma cells (P3×63Ag8.653). Fusedcells were distributed to ten 96-well culture plates (Falcon, Oxnard,Calif.) and grown in hypoxanthine/aminopterin/thymidine (HAT) medium at37° C. with 7.5% CO₂ in a humid atmosphere. Fourteen days later,supernatants were assayed for binding activity to plasma membraneisolate from LNCaP cells and MLD (normal human fibroblasts) using theEnzyme Linked Immunosorbent Assay (ELISA) with anti-mouse IgGβ-galactosidase linked F(ab')₂ fragment from sheep (Amersham Corp.,Amersham, England) or goat anti-mouse IgG horseradish peroxidaseconjugate (Bio-Rad Laboratories, Richmond, Calif.) in a primary screen.Dried membrane isolate (400 ng/well) instead of whole LNCaP cells wasused in the primary screening process because of poor attachment of theLNCaP cells to the plastic wells. To circumvent this problem,immunofiltration on a disposable microfold system (V & P Scientific, SanDiego, Calif.) using whole LNCaP cells was used as a confirmatory assayas described in Section 6.5. In addition, the dot-immunobinding assay onnitrocellulose membrane (Section 6.4) was used to screen thesupernatants of hybridomas for reactivity against LNCaP cell cytosol(100,000×g supernatants) and crude plasma membrane preparation. Todetermine the specificity spectrum of the cultures showing reactivitywith the plasma membranes and/or whole LNCAP cells, the culture fluidswere further tested by ELISA on a panel of an additional 9 viable,normal and neoplastic human cells lines as described in Section 6.5.

6.3. ISOLATION OF PLASMA MEMBRANE-ENRICHED FRACTION

Plasma membrane-enriched fractions were obtained from LNCaP cells andnormal human diploid fibroblast strain MLD by modification of publishedmethods [Kartner, N. et al., J. Membrane Biol. 36:191-211 (1977)].Briefly, MLD cells in roller bottles or LNCaP cells in plastic cultureflasks were gently rinsed 4 times with phosphate buffered saline (PBS).The cells were then rinsed once with hypotonic lysing buffer (3 mM Hepes[hydroxyethylpiperazine-ethanesulfonic acid], pH 7.0, 0.3 mM MgCl₂, 0.5mM CaCl₂) and the buffer discarded. Fresh lysing buffer (5-25 ml) wasadded to each bottle or flask and the cells allowed to swell for 30minutes at room temperature. The swollen cells were removed from thesurface and disrupted by manual shaking. The progress of disruption wasmonitored by phase microscopy of a sample droplet. Gentle trituration(8-10 times) with a 10 ml pipette was used to complete disruption of theLNCaP cells. Vigorous shaking and pipetting were necessary to completelybreak-up the MLD cells. Phenyl-methylsulfonyl fluoride (PMSF, 0.5 mM)[Calbiochem, San Diego, Calif.) was added to minimize proteolysis. Thedisrupted cell suspensions were centrifuged at 100×g to remove nucleiand incompletely disrupted cell clumps. The nuclei pellet was washedonce with the lysing buffer and after centrifugation the supernatant wascombined with the first supernatant and centrifuged at 3,000×g for 10minutes, at 4° C. The pellet consisting of mitochondria and debris wasdiscarded and the supernatant designated as membrane lysate was layeredover a discontinuous density gradient composed of 15 ml each of 10, 30and 38% sucrose (w/v) and centrifuged at 60,000×g for 21/2 hours in anSW 25.2 rotor (Beckman). Material banding at the interface between 10%and 30% sucrose layers was removed by aspiration, washed free of sucroseusing lysing buffer and pelleted by centrifugation at 36,000×g for 60minutes. Pellets were resuspended in PBS and aliquots taken for assay ofprotein and the enzyme phosphodiesterase-I (EC3.1.3.35) as a marker forplasma membranes. The 10/30 plasma membrane isolate was used in thescreening assays for the hybridoma supernatants. All fractions weredispensed and stored as single-use aliquots at -90° C.

6.4. DOT-IMMUNOBINDING ASSAY

The dot-immunobinding assay was used to screen large numbers ofsupernatants of hybridomas producing monoclonal antibodies (Hawkes, etal. Anal Biochem. 119:142-147, 1982) The crude plasma membrane isolate,the 10/30 plasma membrane isolate and/or the cytosol fractionscontaining the cellular antigen were dotted (1-3 μl) on a washednitrocellulose filter paper grid (Bio-Rad, Richmond, Calif.). Theprotein concentration of the "antigen" ranged from between 0.1 to 0.1mg/ml. After thorough drying, the filter was washed in Tris BufferedSaline (TBS, 50 mM Tris-HCl, 200 mM NaCl, pH 7.4). Treatment of thefilter paper with 3% (w/v) bovine serum albumin (Sigma, St. Louis, Mo.)in TBS for 15 minutes at room temperature resulted in the blockage ofnonspecific antibody binding sites on the filter and on the walls of theplastic vessel used to carry out the reaction. The filter paper was thenincubated with hybridoma supernatant or purified monoclonal antibody(2-20 μl ml) for 60 minutes in several changes of TBS, the blocking stepwas repeated. A second antibody [Fab,)2 goat anti-mouse IgG] conjugatedto horseradish peroxidase (Bio-Rad, Richmond, Calif.) (diluted 1:1000 inblocking solution) was added and incubation was carried out at 37° C for120 minutes. After washing with TBS the peroxidase activity wasdeveloped with 4-chloro-1-napthol (0.6 mg/ml in TBS, Merck Inc.) andhydrogen peroxide (0.01% v/v). A positive reaction appeared as a bluecolored dot against the white filter background.

Immunoblotting of cytosol and membrane fractions indicated that thesoluble cytosol fraction of LNCaP cells was not reactive, whilesedimentable (approximately 105,000×g) membrane associated fractionsgave strongly positive spots with monoclonal antibody 7E11.

6 5. ENZYME LINKED IMMUNOSORBENT ASSAY (ELISA)

The enzyme linked immunosorbent assay (ELISA) was used for generalenzyme immunoassay of antigen and screening for monoclonal antibodyproduction. Target cells were seeded (2-30×10⁴ cells/ml) on microtiterplates (Falcon, Oxnard, Calif.) 4-7 days before assay. Nonspecificbinding sites on the plates were blocked with 1% (w/v) swine gelatin ina special media, FL, formulated to keep the cells viable. The FL mediaconsisted of Dulbecco's Modified Eagle Medium (Gibco, Grand Island,N.Y.) supplemented with 15 mM Hepes, 0.3% NaCl, 10 mM NaN₃, and swinegelatin (1% for blocking or 0.3% for washing) pH 7.2-7.4. Hybridomaculture fluids (50 μl per well) were added and incubation was carriedout at 37° C. for 60 minutes. The plates were washed 4 times with FLmedia and F(ab')₂ goat anti-mouse IgG conjugated to horseradishperoxidase (1:1250 in 0.3% swine gelatin, 0.01M PBS, Bio-Rad, Richmond,Calif.) was used in place of the β-galactoxidase conjugate. Afterwashing, 100 μl of substrate (25 ml of 0.1M citrate buffer pH 5.0, 10 μlof 30% H₂ O₂ and 10 mg of α-phenylenediamine, Sigma, St. Louis, Mo.) wasadded to each well. The plate was incubated for 30 minutes in the dark,and the reaction stopped with 50 μl of 2N H₂ SO₄ /well. The absorbancewas determined at 490 nm using the Bio-Tek EIA reader.

Dried crude plasma membrane isolates from LNCaP cells or dried cellswere used initially in the primary screening procedure of hybridomaculture fluids. Approximately 400 ng of membrane protein in 50 μl ofbuffer (S3 mM Hepes, 0.3 mM MgCl₂, 0.5 mM CaCl₂) was dried (35° C.,overnight) in 96 well flat bottom microtirer plates (Falcon, Oxnard,Calif.). Nonspecific binding sites on the plates were blocked with 1%swine gelatin in PBS containing 0.1% NaN₃. With the exception of thewash buffer consisting of 0.01M Hepes and 0.2 μM of PMSF in saline pH7.6, all other reagents used were as described above for cell surfaceenzyme immunoassay.

LNCaP cells attach poorly to plastic wells and detach from the plasticsurface during the ELISA procedure. To circumvent this problemimmunofiltration on a disposable microfold system (V & P Scientific, SanDiego, Calif.) using whole LNCaP cells was employed as a confirmatoryassay for the dried membrane assay. After nonspecific binding sites onthe disposable microfold system was blocked with 5% human serum albumin(HSA) in PBS, 2.5×10⁴ LNCaP cells in 100 μl of 5% HSA buffer wasdeposited on the filter discs with vacuum. After washing the filterswith 0.3% gelatin in 0.01M phosphate buffer, the plates were processedas described above. However, after incubation with substrate, thereaction mixture from each well (100 μ) was transferred to 1/2 areaCostar plates (Costar, Cambridge, Mass.) before spectrophotometricdetermination on the BIO-TEK EIA reader. Hybridomas were detected inapproximately 500 culture wells. 206 hybridomas were successfullyexpanded and on primary ELISA screen, 126 reacted with partiallypurified LNCaP membranes, 92 reacted with intact LNCaP cells 76 reactedwith normal human fibroblast-cells and membrane preparations. Furtherscreening by ELISA and by immunoperoxidase staining on a panel ofadditional 11 viable, normal and neoplastic cell lines and byimmunoblotting of cytosol and membranes fractions from LNCaP cellsnarrowed the field of 2 cloned hybridoma cell lines of particularinterest, including MoAb 7E11 and 9H10.

Hybridoma cultures showing specificity restricted to the LNCaP cells andmembranes were cloned by limiting dilution and subcloned in agarose [seee.g., Schreier, M. et al., Hybridoma Techniques, pp. 11-15, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1980)]. Stable cultures ofantibody-producing hybridomas were expanded in complete media [RPMI 1640media supplemented with 10% (w/v) heat-inactivated fetal bovine serum,100 U/ml penicillin, 100 μg/ml streptomycin, and 10 μg/ml insulin(GIBCO) and cryopreserved. After cloning, two stable monoclonalhybridoma cell lines were obtained and designated as 7E11-C5 and 9H10-A4respectively.

Exhausted culture fluids and mouse ascites fluids were the source ofantibodies used for further studies. Ascites fluid from mice carryingthe hybridoma cell line was used to obtain large quantities ofmonoclonal antibodies. Hybridoma cells for ascites fluid production werewashed 2 times with RPMI 1640 medium and resuspended at a density of1-5×10⁷ cells/ml. Using a 20-gauge needle, 0.2 ml of the cell suspensionwas injected into the peritoneal cavity of female nude mice. Pristanewas not routinely used to precondition the animals. Ascites fluidcontaining high titers of antibodies was regularly harvested 4-5 weeksafter injection with the hybridoma cells.

6.6. ISOTYPING OF MONOCLONAL ANTIBODIES

Monoclonals 7E11-C5 and 9H10-A4 are of the IgGl subclass, as determinedby double diffusion gel precipitation with isotype specific antisera(Miles). Consistent with this finding were observations that Protein Aconjugated with either fluoroscein or horseradish peroxidase (BIO-RAD)failed to react with smears of LNCaP cells following incubation witheither monoclonal.

6.7. INDIRECT IMMUNOPEROXIDASE STAINING OF TUMOR

SPECIMENS OF MONOCLONAL ANTIBODIES 7E11C5

In a first set of experiments, cytospin smears of cultured cells,formalin-fixed cryostat (-25° C.) sections and sections of formalinfixed, paraffin embedded human tissues were used for immunoperoxidasestaining as described previously [Heyderman E. and Neville, A. M., J.Clin. Path., 30:138-140 (1976)]. Briefly, hydrated paraffin tissuesection or formalin fixed cryostat sections and cytospin smears (2%paraformaldehyde, pH 7.5, 60 minutes, at room temperature) were treatedwith 10% pooled normal human serum in PBS and washed for 15 minutes inPBS containing 0.5% Nonidet P-40. The sections were then incubated (60min., 37° C.) with monoclonal antibody preparations (30 μl, 5-20 μg/ml)diluted in PBS containing 1% w/v BSA (Sigma). Following 4 washes in PBS,peroxidase conjugated goat antibodies against murine immunoglobulin(1:50 dilution in bovine serum albumin (Bio-Rad) were applied for 60minutes at 37° C. After thorough washing in PBS (4 changes), theperoxidase activity was revealed using diaminobenzidine 0.5 mg/ml): H₂O₂ (0.01%) substrate in 0.1M Tris buffer pH 7.2 The sections weredehydrated in increasing concentrations of ethanol to xylene and thenmounted in Permount (Fisher Scientific, Fairlawn, N.J.). In addition tothe normal controls, control experiments which included PBS in place ofthe primary antibody, peroxidase-conjugated antibody alone and culturefluid from the myeloma cell line P3×63Ag8.653 were used. The intensityof the immunoreaction product was evaluated using a Zeiss microscope(40×objective; 10×ocular), and scored using a(-) to (+++) scale.

The reactivity of the two stable monoclonal antibodies with LNCaP cells,as well as 32 other human normal and malignant cell lines, is presentedin Table I.

                  TABLE I                                                         ______________________________________                                        REACTIVITY OF MoAb 7E11-C5 AND MoAb 9H10-A4                                   WITH CULTURED HUMAN CELLS BY ELISA                                            AND IMMUNOPEROXIDASE STAINING                                                                      Reactivity with                                                                 MoAb     MoAb                                          Human Cells in Culture 7E11-C5  9H10-A4                                       ______________________________________                                        LNCaP     Prostatic Ca     +++      ++++                                      DU145     Prostatic Ca     --       --                                        PC-3      Prostatic Ca     --       --                                        RT-4      Bladder Ca       --       --                                        5637      Bladder Ca       --       --                                        MCF-7     Breast Ca        --       --                                        MDA-MB-231                                                                              Breast Ca        --       --                                        HT-29     Colon Ca         --       --                                        SK        Colon Ca         --       --                                        COLO205   Colon Ca         --       --                                        PAC       Pancreatic Ca    --       --                                        TT        Medullary Thyroid Ca                                                                           --       --                                        MeWo      Melanoma         --       --                                        SM        Melanoma         --       --                                        HeLa-531  Uterine Ca       --       --                                        HeLa-CCL2 Uterine Ca       --       --                                        A209      Rhabdomysarcoma  --       --                                        SW872     Liposarcoma      --       --                                        HT1080    Fibrosarcoma     --       --                                        5959      Osteogenic Sarcoma                                                                             --       --                                        SAOS-2    Osteogenic Sarcoma                                                                             --       --                                        HBC       Bronchogenic Ca  --       --                                        A549      Lung Adeno Ca    --       --                                        CHAGO     Large Cell Lung Ca                                                                             --       --                                        SKMES     Squamous Cell Lung Ca                                                                          --       --                                        PC-1      Lung Ca          --       --                                        PC-9      Lung Ca          --       --                                        PC-14     Lung Ca          --       --                                        T-24      Lung Ca          --       --                                        MLD       Normal Fibroblasts                                                                             --       --                                        BG-9      Normal Fibroblasts                                                                             --       --                                        GM2504    Normal Fibroblasts                                                                             --       --                                        FL        Human Amnion     --       --                                        ______________________________________                                    

The indirect immunoperoxidase staining of formalin fixed LNCaP cells bysupernatants from either of the hybridoma cultures was positive indilutions ranging from 1:200 to 1:800 while ascitic fluids harvestedfrom mice stained LNCaP smears at dilutions from 1:50,000 to 1:400,000.The localization of immunoperoxidase staining of LNCaP cells differedfrom MoAb 7E11-C5 and MoAb 9H10-A4. MoAb 7E11-C5 staining was apparentover the cytoplasmic region with intensity slightly increasing towardthe cell periphery (FIG. 2) MoAb 9H10-A4 produced continuous, narrowband of strong staining associated with the cell periphery (FIG. 3). Thestaining pattern of LNCaP cells from culture, as well as cells takendirectly from nude mouse tumors was constant for each MoAb.

Antigen reactive with MoAb 7E11-C5 was best preserved by neutralformalin or cold acetone fixation. Methanol, ethanol, propanol andchloroform reduced reactivity: ethyl ether, Nonidet (0.1%) and TritonX-100 were without effect. Drying and storage at -80° for more than 3days, regardless of fixation, as well as treatment with periodate orBouin's fluid, destroyed the 7E11-C5 antigen, suggesting its sensitivityto oxidation. Specimens embedded in paraffin after formalin fixationretained only small fraction of the original reactivity. Antigendetected by MoAb 9H10-A4 appears to be more stable and was fullyreactive after exposure to all tested fixatives, as well as afterprolonged storage.

Viable LNCaP cells when stained by the indirect immunofluorescencemethod showed bright peripheral rings after exposure to MoAb 9H10-A4. Nostaining of viable cells, however, was seen with MoAb 7E11-C5.

None of the 32 other cell lines (normal or malignant) showed anyreactivity with either ELISA or indirect immunoperoxidase staining,regardless of fixation.

In an expanded analysis of the distribution of the epitope recognized bythese two antibodies, indirect immunoperioxidase staining of frozensections after fixation in neutral formaldehyde was performed. Freshfrozen sections of human normal and neoplastic tissues obtained frombiopsy, surgery and autopsy were fixed in 2% neutral formaldehyde.Results from observations made on 175 specimens are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                        ANTIGEN IN FROZEN SECTIONS FROM 175 SPEC-                                     IMENS DETECTED BY INDIRECT IMMUNO-                                            PEROXIDASE STAINING WITH MoAbs 7E11-C5 AND                                    9H10-A4                                                                                          Positive/Total Tested                                                         MoAb   MoAb                                                                   7E11-C5                                                                              9H10-A4                                                                Reactive                                                                             Reactive                                            ______________________________________                                        Human Prostatic Epithelium                                                    CaP foci in prostate 9/9      0/9                                             CaP metastases in lymph nodes                                                                      2/2      0/2                                             Benign prostatic hypertrophy                                                                       5/7      0/7                                             Normal prostates     9/9      0/9                                             Human Tumors (Non-Prostatic)                                                  Breast Ca            0/8      0/8                                             Renal                0/3      0/3                                             Bladder Ca           0/2      0/2                                             Adrenal Ca           0/2      0/2                                             Colon Ca             0/2      0/2                                             Sarcoma              0/2      0/2                                             Squamous Cell Ca     0/3      0/3                                             Melanoma             0/1      0/1                                             Neuroblastoma        0/1      0/1                                             Uterine Ca           0/1      0/1                                             Pancreatic Ca        0/1      0/1                                             Normal Human Organs                                                           Urinary Bladder      0/5      0/5                                             Ureter               0/5      0/5                                             Seminal Vesicles     0/3      0/3                                             Testis               0/4      0/4                                             Kidney                2/14     0/14                                           Ovary                0/3      0/3                                             Uterus               0/3      0/3                                             Breast               0/3      0/3                                             Bronchus             0/4      0/4                                             Lung                 0/5      0/5                                             Liver                0/7      0/7                                             Spleen               0/8      0/8                                             Pancreas             0/5      0/5                                             Tongue               0/2      0/2                                             Esophagus            0/1      0/1                                             Stomach              0/3      0/3                                             Colon                0/8      0/8                                             Thyroid              0/5      0/5                                             Parathyroid          0/1      0/1                                             Adrenals             0/4      0/4                                             Lymph Node           0/5      0/5                                             Skeletal Muscle      0/5      0/5                                             Heart                0/5      0/5                                             Aorta                0/3      0/3                                             Vena Cava            0/3      0/3                                             Brain                0/1      0/1                                             Skin                 0/4      0/4                                             ______________________________________                                    

MoAb 7E11-C5 stained both malignant and apparently normal prostaticepithelial cells with remarkable selectivity. No reactivity was seen instromal components such as fibers, vessels, muscles, etc. Positive cellsstained stronger toward the cell periphery. The staining showed a smalldegree of heterogeneity among individual cells. A difference was notedin the intensity of staining between normal and neoplastic epithelium.The staining of CaP cells was strong in 9 out of 11 specimens and ofmoderate intensity in the remaining 2. Apparently normal andhypertrophic prostatic glands showed faint (in 12 out of 16 specimens)to moderate (2 out of 16) staining. Two specimens from benign prostatichypertrophy (BPH), which were classified as negative, contained onlyvery few rudimentary structures reminiscent of prostatic ducts. Overall,25 out of 27 specimens from prostates and CaP reacted with MoAb 7E11-C5.

Despite strong staining of cytoplasmic membranes of LNCaP cells, MoAb9H10-A4 failed to react in frozen sections with either normal prostaticepithelium or with neoplastic cells.

Neither MoAb 7E11-C5 nor MoAb 9H10-A4 stained fresh frozen sections fromany of the 26 specimens representing 11 different histological types ofhuman non-prostatic tumors.

Among 122 individual specimens from 28 different normal human organs andtissues, 120 did not show any staining with MoAb 7E11-C5. In 2 instances(out of 14) of normal kidneys, poorly defined, low intensity, diffuseand uneven brownish deposits were detected on the inner surfaces and inthe lumen of some of the Henle's loops. Pre-incubation of fixed sectionswith 1% albumin or gelatin solutions reduced such "staining". Similarreactions in the human kidney by the immunoperoxidase staining withvarious murine monoclonal antibodies were noted by other authors, butthe significance, if any, or the specificity of such "staining" is atpresent unclear. Again, MoAb 9H10-A4 did not react with any of the 122specimens from normal organs.

6.8. COMPETITIVE BINDING ELISA

After incubation of MoAb 7E11-C5 at appropriate concentrations (20-100ng/ml) with whole LNCaP cells, hypotonic cell lysates, LNCaP cellsonicates or partially purified cell membranes, the original activity ofMoAb 7E11-C5 as measured by ELISA was significantly and reproduciblyreduced. The inhibition was a function of antigen concentration and thelength of incubation time (results not shown). These observationssuggested that MoAb 7E11-C5 reactive antigen could also be detected, ifpresent, in human sera using appropriately designed assay.

Initial experiments were focused on the assay specificity andmethodology. For these studies, 3 sera from CaP patients inhibiting MoAb7E11-C5 in competitive binding ELISA were used. Centrifugation (2 hrs;100,000×g) failed to sediment their inhibitory activity which suggestedthat the "inhibitor" in serum is not associated with circulating wholeCaP cells, membrane vesicles or cell fragments, but represents the MoAb7E11-C5 reactive epitope in a soluble form. This observation wasunexpected since high speed centrifugation of either disrupted LNCaPcells, or spent LNCaP cell culture media yields anti-MoAb 7E11-C5directed reactivity only in sedimentable fractions, indicating that theMoAb 7E11 specific epitope is associated with insoluble supramolecularaggregates. The level of competitive binding ELISA inhibitory activityagainst MoAb 7E11-C5 in human sera remained constant after 10 cycles ofrepeated freezing and thawing, heating to 56° for 30 min., 6 monthsstorage at -80°, as well as after overnight incubation at 37° regardlessof addition of protease inhibitors.

ELISA inhibitory activity was not due to the presence in tested sera ofa human antibody with specificity similar to MoAb 7E11-C5, which couldcompetitively block available antigenic sites on the LNCaP detectorcells, nor were enzymatic activities of serum affecting the antigenicsites of LNCaP cells. This was shown by preincubation (up to 72 hrs.) ofwells containing LNCaP cells with either "inhibitory" serum,non-inhibitory serum or PBS. The serum was then removed and MoAb 7E11-C5activity was tested by standard ELISA procedure. No reduction inreaction intensity was observed between control wells and wellspre-incubated with inhibitory sera.

In addition, either the presence in sera of anti-murine IgG capable ofbinding MoAb 7E11-C5 or the existence of an unusual proteolytic activitydirected against monoclonal antibodies in general, was excluded bypre-incubation of inhibitory sera with murine MoAb 9H10-A4 and showingthat immunologic reactivity with LNCaP cells and membranes wasunaffected.

Next, the possibility was investigated that "inhibitors" in positive CaPsera were unspecific and interacted only with the Fc portion ofMoAb7E11. To this end, the inhibition of immunoreactivity of 7E11F(ab')₂ antibody fragments by CaP sera was tested. The F(ab')₂ antibodyfragments were as susceptible to inhibition by positive human sera fromCaP as were the complete MoAb 7E11-C5.

Taken together, the above experiments indicate that observed ELISAinhibition results from specific immunological reaction between MoAb7E11and corresponding antigen present in serum from some CaP patients.

The assay methodology for testing human sera from normal blood donors,non-prostatic malignancies and patients with prostatic cancer forspecific binding of MoAb 7E11-C5 in limiting concentrations wasestablished as follows:

Aliquots (125 μl) of serum were incubated (3 hrs., room temp.) with:

a) 125 μl of diluent (PBS with 0.3% bovine serum albumin, pH 7.2, sodiumazide 0.05%)

b) 125 μl of MoAb 7E11(60 ng/ml in diluent)

c) 125 μl of MoAb 9H10 (6 ng/ml in diluent).

As references of total MoAb activity in the absence of serum, MoAb7E11-C5 (30 ng/ml) and MoAb 9H10-A4 (3 ng/ml) in diluent only were used.In addition, each microtiter plate contained a set (12 wells) ofexternal controls consisting of normal female serum preincubatedseparately with each MoAb and diluent.

The reaction mixtures were then incubated in a single 96 well microtiterplate (Falcon) overnight (18 hrs, 4° C.; quadruplicate wells, 50μl/well) with air dried LNCaP cells (4×10⁴ cells/well, 2.0% formaldehydefixed for 30 min) to determine reactivity by ELISA. The results of theELISA test (O.D. read at 490 nm) are expressed as the SpecificReactivity with MoAb 7E11-C5 factor (SR7E11 factor). The SR7E11 factoris calculated according to the formula: ##EQU1##

The inclusion of MoAb 9H10 in the test allows to compensate forpotential differences in kinetics of binding of MoAb to target LNCaPcells in high (50%) serum concentration, as well as for unexpectedpresence in individual sera of interfering macromolecules (anti-murineIgG, enzymes, etc.). The MoAb 9H10-A4 strongly binds to LNCaP plasmamembranes, but is unrelated in specificity to MoAb 7E11-C5 and does notreact with other human cell lines, or frozen sections of normal humanorgans or malignant tumors. Neither normal nor CaP sera inhibitspecifically MoAb 9H10-A4.

To examine the kinetics of SR7E11 factor changes, normal control andpositive CaP sera were preincubated with MoAbs 7E11-C5 and 9H10-A4 forperiods ranging from 3 hrs. up to 10 days. The antigen-antibody reactionwas thus allowed to proceed to or near completion at limiting antibodyconcentrations. Table 3 shows that the SR7E11 factor of positive CaPsera significantly increases during prolonged serum-MoAb incubation,while SR_(7E11) of control sera remains low or even decreases. Thisfurther supports the notion that SR_(7E11) factor reflects the amount ofantigen in serum binding MoAb 7E11. Another explanation is alsoplausible: sera from prostatic cancer patients could containantiidiotypic antibodies (anti-Id) of the Ab-2 variety. Such antibodiescould carry specificity and reactivity similar to the epitope associatedwith insoluble membranes of LNCaP cells, i.e., the epitope against whichthe murine monoclonal 7E11-C5 is directed. Therefore, the anti-Id couldbind to 7E11-C5 and be the cause of positive results in the competitiveinhibition ELISA in CaP serum.

                  TABLE 3                                                         ______________________________________                                        CHANGES IN SR.sub.7E11 FACTOR AS A FUNCTION OF                                PRE-INCUBATION TIME OF MoAbs WITH HUMAN                                       SERA                                                                                     Time of Pre-incubation with MoAbs                                  Serum Source 3 Hrs.    3 Days  5 Days                                                                              10 Days                                  ______________________________________                                        Normal Female                                                                              1.28      1.33    0.98  1.19                                     Normal Male  1.14      1.32    1.21  1.09                                     Pool of Young Males                                                                        1.19      1.43    1.28  0.99                                     Prostatic Ca N° 1                                                                   2.07      4.11    6.83  7.34                                     Prostatic Ca N° 2                                                                   3.07      10.64   14.30 15.95                                    ______________________________________                                    

To establish the average numerical value of SR_(7E11) factor for normal,healthy individuals, 30 sera from RPMI Blood Bank donors were tested.The mean SR_(7E11) of this group was 1.13±0.23 (x±S.D.). No significantdifferences between the mean values of the SR_(7E11) factor for groupsof males and females were found. For the threshold defining positiveresults (at the p<0.01 level), x⁻ +3 S.D. was calculated to be 1.82. Thevalue above 1.82 for SR_(7E11) was used for the classification ofSpecific Reactivity as positive.

Subsequently, additional 116 sera were tested: 43 from CaP patients, 7from individuals with benign prostatic hypertrophy and 66 sera fromnon-prostatic malignancies. Tables, 4, 5 and 6 show the results. Astrong statistical correlation emerged between the assay positiveoutcome and diagnosis of prostatic cancer (Table 4). In addition, asshown in Table 5, the patients with positive SR_(7E11) were more likelyto be in progression than those who were negative. Similarly, a higherpercentage of positive tests were among patients with widelydisseminated disease vs. less advanced clinical stages. Among 66 serafrom individuals with tumors of non-prostatic origin, only 3 (4.6%)tested positive (Table 6). Two of the positive sera were from femaleswith disseminated uterine and renal carcinomas respectively. The thirdpositive serum was obtained from young male with testicular embryonalcarcinoma.

                  TABLE 4                                                         ______________________________________                                        SUMMARY TABLE OF MoAb 7E11-C5 COMPETITIVE                                     BINDING ELISA IN HUMAN SERA                                                   Serum Source     Number Tested                                                                             SR.sub.7E11 Positive                             ______________________________________                                        Prostatic Cancer (CaP)                                                                         43          20     (46.5%)                                   Benign Prostatic  7          0                                                Hypertrophy (BPH)                                                             Non-Prostatic Malignancies                                                                     66          3      (4.6%)                                    Normal Blood Donors                                                                            30          0                                                Total            146         23                                               ______________________________________                                    

Two tail Fisher Exact Probability Test indicates that there is asignificantly higher SR_(7E11) positive rate (p<0.0001) in a populationof 43 CaP patients as opposed to a group of 103 non-CaP controls(normal, BPH and other malignancies). The assays were blinded.

                  TABLE 5                                                         ______________________________________                                        MoAb 7E11-C5 COMPETITIVE BINDING ELISA                                        IN PROSTATIC CANCER                                                           Clinical Evaluation                                                                          Number Tested SR.sub.7E11 Positive                             ______________________________________                                        No Apparent Disease                                                                           7            0                                                Remission/Stable                                                                             13            6      (46%)                                     Progression    23            14     (61%)                                     Total          43            20                                               CaP Stage                                                                     B I             2            0                                                B II            5            1      (29%)                                     C I             3            2                                                D I             4            1                                                D II           29            16     (55%)                                     Total          43            20                                               ______________________________________                                    

Logistic regression relating the probability that the patient was in CaPprogression to the SR_(7E11) indicates a significant (at p<0.05)relationship. Patients with positive SR_(7E11) are more likely to be inprogression, than those who are negative. The assays were blinded.

                  TABLE 6                                                         ______________________________________                                        MoAb 7E11-C5 COMPETITIVE BINDING ELISA IN                                     HUMAN SERA FROM NON-PROSTATIC                                                 MALIGNANCIES                                                                  Diagnosis         SR.sub.7E11 Positive/Total Tested                           ______________________________________                                        Testicular Tumors (Seminoma,                                                                     1/16                                                       Embryonal Ca, Teratoma)                                                       Transitional Cell Ca (Bladder)                                                                  0/7                                                         Renal Cell Ca      1/4*                                                       Breast Ca         0/3                                                         Ovarian Adeno Ca  0/3                                                         Uterine Adeno Ca   1/2*                                                       Gastric Ca        0/3                                                         Hepatoma          0/2                                                         Pancreatic Adeno Ca                                                                             0/3                                                         Colon and Rectum Adeno Ca                                                                       0/3                                                         Lung Ca           0/3                                                         Sarcoma           0/4                                                         Astrocytoma, Chordoma                                                                           0/2                                                         Squamous Cell Ca  0/3                                                         Basal Cell Ca     0/2                                                         Histiocytoma      0/1                                                         Mesothelioma      0/1                                                         Lymphoma, Leukemia                                                                              0/4                                                         Total              3/66      (4.6%)                                           ______________________________________                                         *SR.sub.7E11 positive sera were from terminal patients who expired shortl     after testing.                                                           

MoAb 7E11-C5 and MoAb 9H10-A4 were of the IgGl subclass and as such,either alone or with complement, lacked detectable biological activitiesagainst LNCaP cells in vitro or in nude mice. Both MoAbs reacted inELISA and by immunoblotting with sedimentable, cytoplasmic membrane richfractions of LNCaP cells, but not with soluble cytosol or secretoryglycoproteins such as PSA or PAP. The antigen with which 7E11-C5 reacts,in cultured LNCaP cells, is strictly associated with non-soluble,sedimentable material. In contrast, many CaP patients, serum containssuch epitopes in soluble form.

MoAb 9H10-A4 had specificity restricted to epitopes present on thesurface of LNCaP cell plasma membrane as demonstrated by ELISA andimmunospecific staining of a variety of viable or fixed cells and frozensections. No binding of MoAb 9H10-A4 was detected to any other thanLNCaP human prostatic and non-prostatic normal or malignant cells instudies involving 32 cell lines, 27 prostates and 148 other fresh-frozenspecimens of human organs, normal tissues and tumors. This suggests thatMoAb 9H10-A4 defined antigen could be unique for an individual prostatictumor or perhaps even a single metastasis from which the LNCaP cellswere isolated. However, since our study involved only 18 specimens fromCaP, all of which were negative, an additional possibility remains thatMoAb 9H10-A4 could be detecting epitopes which are associated with arapidly progressing in younger males rare form of CaP from which LNCaPcells were originally isolated. Before these speculations areexperimentally tested, MoAb 9H10-A4 remains as a useful reagent topositively identify LNCaP cells and distinguish them from other culturedcells. In addition, this MoAb serves as a reliable control incompetitive binding ELISA with MoAb 7E11-C5 for detection of circulatingantigens associated with CaP.

MoAb 7E11-C5 reacted with epithelial cells in frozen sections fromprostatic carcinoma, benign prostatic hypertrophy and to a lesser degreewith normal prostatic glands. Among 33 grown in vitro normal andneoplastic cell lines, only LNCaP cells bound MoAb 7E11-C5 in ELISA andin indirect immunospecific staining of dried and fixed smears. It is ofinterest that CaP derived DU-145 and PC-3 cells did not exhibit anyreactivity with MoAb 7E11-C5. This finding parallels the absence ordiminution of phenotypic expression in PC-3 and DU-145 of other markermolecules (PAP, PSA, androgen receptors) which are characteristic ofhuman epithelial prostatic cells, and are abundantly preserved in LNCaPcultures (Kaighn, et al., in Models for Prostatic Cancer, pp. 85-109,Alan R. Liss, Inc., N.Y., 1980; Kaighn et al., Invest. Urol. 17:16-23,1979; Papsidero et al., Cancer Res. 40:3032-3035, 1980; Stone et al.Int. J. Cancer 21:2374-281, 1978). Strong reactivity of MoAb 7E11-C5with LNCaP membrane preparations and fixed cells contrasted sharply withthe lack of staining by the indirect immunofluorescence method ofviable, unfixed LNCaP cell suspensions. This observation suggests thatepitopes specific for MoAb 7E11-C5 are either absent or not availablefor binding on the outer surface of living LNCaP cells. It remains to bedetermined whether such restriction applies to normal and malignantviable cells from human prostates. The results of such experiments couldhelp to project the practical potential of appropriate MoAb 7E11-C5conjugates as either imaging or therapeutic agents for CaP.

The evidence for selective specificity of MoAb 7E11-C5 for humanprostatic epithelium was reinforced by consistently negative results ofimmunospecific staining of numerous fresh frozen sections from a widerange of human non-prostatic normal or malignant tissues. Noted on acouple of occasions, poorly defined staining of kidney tubules requireadditional observations to ascertain its reproducibility and specificityon a larger size sample of fresh biopsy specimen.

Cell lines 7E11-C5 and 9H10-A4 as described herein have been depositedwith the American Type Culture collection, Rockville, Md., and have beenassigned ATCC designation HB 10494 and ATCC Safe Deposit DesignationS.D. No. 1308, respectively. The invention described and claimed hereinis not to be limited in scope by the cell line deposited, since thedeposited embodiment is intended as a single illustration of one aspectof the invention and any equivalent cell lines which produce afunctionally equivalent monoclonal antibody are within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims.

What is claimed is:
 1. A monoclonal antibody produced by hybridoma cellline 7E11-C5, ATCC Designation HB 10494, which monoclonal antibody bindsspecifically to an epitope present on a membrane associated antigen ofhuman prostatic cancer epithelium and normal prostatic epithelium andwhich does not bind to non-prostatic antigens present in other tissues.2. Hybridoma cell line 7E11-C5, ATCC Designation HB 10494.